As described in the goals and objectives section of this report, this project consists of four specific aims:[unreadable] [unreadable] Developing Tools for Experimental Analysis of Vascular Development in the Zebrafish[unreadable] [unreadable] This has been an important aim of this project. We previously established a microangiographic method for imaging patent blood vessels in the zebrafish and used this method to compile a comprehensive staged atlas of the vascular anatomy of the developing fish (http:eclipse.nichd.nih.govnichdlmgredirect.html). We have also generated a variety of transgenic zebrafish lines expressing different fluorescent proteins within vascular or lymphatic endothelial cells, making it possible for us to visualize vessel formation in intact, living embryos. We have developed methodologies for long-term multiphoton confocal timelapse imaging of vascular development in transgenic fish, and used these methods to examine blood vessel patterning and lumenogenesis, and the ontogeny of the lymphatic system. The recent development of Tol2-transposon based vectors for transgenesis in zebrafish has greatly facilitated generation of new transgenic lines, and we are continuing to develop many new lines useful for in vivo vascular imaging as well as for in vivo endothelial-specific functional manipulation of signaling pathways involved in vascular specification, patterning, and morphogenesis.[unreadable] [unreadable] Genetic Analysis of Vascular Development[unreadable] [unreadable] We use forward-genetic approaches to identify and characterize new zebrafish mutants that affect the formation of the developing vasculature. We are carrying out an ongoing large-scale genetic screen for ENU-induced mutants using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels. We have screened well over 2000 genomes to date, and identified over 100 new vascular mutants with phenotypes including loss of most vessels or subsets of vessels, increased sproutingbranching, and vessel mispatterning. A bulked segregant mapping pipeline is in place to rapidly determine the rough position of newly identified mutants on the zebrafish genetic map, and fine mapping and molecular cloning is in progress for many mutants. We have already positionally cloned the defective genes from a number of vascular-specific mutants, including violet beauregarde (defective in Alk1acvrl1), plcg1 (defective in phospholipase C-gamma 1), cds2 (defective in phosphoinositol signaling), kurzschluss (defective in a novel chaperonin), beamter (defective in trunk somite and vascular patterning), etsrp (an ETS-related transcription factor). Ongoing mutant screens and positional cloning projects in the lab are continuing to yield a rich harvest of novel vascular mutants and genes, bringing to light new pathways regulating the formation of the developing vertebrate vasculature.[unreadable] [unreadable] Analysis of Vascular Morphogenesis[unreadable] [unreadable] Classical studies dating back more than 100 years suggested a model for assembly of endothelial tubes via formation and fusion of vacuoles, but conclusive in vivo evidence for this model was lacking, primarily due to difficulties associated with imaging the dynamics of sub-cellular endothelial vacuoles deep within living animals. Taking advantage of the favorable optical properties of the fish and novel transgenic lines that we have developed, we used high-resolution time-lapse two-photon imaging to show that the formation and intra- and inter-cellular fusion of endothelial vacuoles drives vascular lumen formation. We are currently developing transgenic lines that permit us to visualize the dynamics of endothelial cell-cell junctions and intracellular cytoskeletal structures to examine their role in the cellular rearrangements that occur during vascular sprouting and growth and vascular tube formation. At the same time, we are studying a number of different genes required for vascular morphogenesis and vascular integrity, including pak2a and rap1b, in order to begin to dissect the molecular regulatory mechanisms controlling vascular morphogenesis and the maintenance of vascular integrity.[unreadable] [unreadable] Analysis of Vascular Patterning[unreadable] [unreadable] We have used multiphoton time-lapse imaging to characterize patterns of vessel assembly throughout the developing zebrafish, and ongoing studies in the laboratory are aimed at understanding how this pattern arises and the what cues guide vascular network assembly during development. We previously demonstrated that known neuronal guidance factors play an important previously unknown role in vascular guidance and vascular patterning, showing that Semaphorin signaling it is an essential determinant of trunk vessel patterning. Current studies are aimed at further understanding the role of additional factors guiding the patterning of developing vascular networks in vivo, the role of matrix proteins in vessel growth in vivo, and the role of local organs and tissues in helping guide vessel patterning.